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Leucocytic infiltration into the hypoglossal nucleus following injury to the hypoglossal nerve.

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Leucocytic Infiltration into the Hypoglossal Nucleus
Following Injury to the Hypoglossal- Nerve '
E. K. ADRIAN, JR.,2 AND R. D. SMOTHERMON
Department of Anatomy, The University of Texas Medical Branch,
Galveston, Texas 77550
ABSTRACT
Tritiated thymidine was injected into adult female A/J mice at 24,
20, and 16 hours before transection of the left hypoglossal nerve. These animals and
uninjured controls that received the same sequence of tritiated thymidine injections
were sacrificed by perfusion fixation a t either 4, 8, 16 or 35 days after the time of
the injury. At all time intervals after injury there were several times more labeled
cells in the nucleus of the injured nerve than were found in the right hypoglossal
nucleus of the same animals. However, the right hypoglossal nucleus of the injured
animals had significantly more labeled cells than were found i n the hypoglossal nuclei
of the uninjured control animals. Labeled mitotic figures were present in the nucleus of
the injured nerve o n the fourth day after injury. Since no tritiated thymidine should
have been available a t the time of injury or afterward to label cells that proliferated in
response to the injury, the difference in the number of labeled cells between the
injured animals and the controls is attributed to a n infiltration of labeled mononuclear leucocytes.
Recently a number of authors have de- nervous tissue wounds has been repeatedly
monstrated proliferation of cells closely as- demonstrated (Adrian and Walker, '62;
sociated with the cell bodies of fibers that Walker, '63; Konigsmark and Sidman, '63;
have been damaged in peripheral nerve Smith and Walker, '67; Adrian, '68). These
injuries (Cammermeyer, '65a,b; Sjostrand, cells can synthesize DNA and undergo mi'65a,b; Watson, '65; Kreutzberg, '66; totic division after entering nervous tissue
Friede and Johnstone, '67; Humbertson, (Adrian, '68). The experiment to be de'68). These proliferating cells have usu- scribed demonstrates the presence of large
ally been referred to as glial satellites, al- numbers of cells considered to be monothough Cammermeyer ('65a) called them nuclear leucocytes in the hypoglossal nujuxtavascular histiocytes. Following the cleus following injury to the hypoglossal
period of greatest proliferative activity in- nerve.
creased numbers of cells identified as miMATERIALS AND METHODS
croglia have been observed (Rap05 and
In an initial experiment four adult feBako;, '59; Cammermeyer, '65a; Sjostrand,
'66). Since the cells in question were male A/J mice weighing 25-30 gm were
labeled when tritiated thymidine was in- each given three subcutaneous injections
jected shortly before sacrifice and since of tritiated thymidine, 2 pc per gm body
mitotic figures were present, there seems weight, at four-hour intervals. Twentyto be no question that DNA synthesis and four hours after the first injection each
mitotic divisions occur. Also, the published animal was anesthetized with amobarbital
photographs of mitotic figures in a juxta- sodium, 0.16 mg per gm body weight given
vascular position and of labeled cells that intraperitoneally in 5% solution. The venare clearly satellites to neurons leave little tral neck region was shaved, and a middoubt as to the places where these cells are line incision was made from the manufound. That the observed numerical in- brium to the mandible. The left hypogloscrease in non-neuronal cells associated sal nerve was dissected out and divided at
with injured neurons is due entirely to
Received May 27, '68. Accepted July 25. '69.
proliferation of permanent inhabitants of
1 This investigation was supported by PHS Research
No. FR 05427 and bv the McLaughlin Faculty
the nervous tissue is, however, open to Grant
Fund for Preliminary Research.
2 Present address:
Department of Anatomy, The
question. The presence of large numbers
University of Texas Medical School at San Antonio,
of mononuclear leucocytes in and around 7703
Floyd Curl Drive, San Antonio, Texas 78229.
~~
ANAT. REC.,166: 99-116.
99
100
E. K. ADRIAN, JR. A N D R. D. SMOTHERMON
a point distal to where the descending
ramus is given off and proximal to the
branches to the extrinsic muscles of the
tongue. The nerve was divided by grasping
it with two pairs of fine forceps and pulling it apart. The skin wound was then
closed with 6-0 silk sutures, and the
animal was allowed to recover. Four days
following division of the nerve, the animals
were again anesthetized with amobarbital
and were sacrificed by intracardiac perfusion with 4% phosphate buffered formaldehyde prepared as described by Pease
('64).
In a second experiment the dosage of
tritiated thymidine was doubled. Eleven
adult A/J female mice were each given
three subcutaneous injections of tritiated
thymidine, 4 pc per gm body weight, at
four-hour intervals. In seven of these
animals the left hypoglossal nerve was injured 16 hours after the last injection of
tritiated thymidine in the manner already
described. The remaining four animals
were left as uninjured controls. Two injured animals and one uninjured control
were sacrificed by intracardiac perfusion
with 4% phosphate buffered formaldehyde
4, 8, or 35 days later. One injured animal
and one uninjured control were sacrificed
in the same manner 16 days after the nerve
injury.
Following the perfusion the skin covering the head of each animal was cut in the
midline, and the calvarium was opened
without disturbing the brain. The whole
animal was then immersed in fixative prepared in the same way as that used for the
perfusion.
After remaining in fixative for at least
one night, the medullary parts of the brain
stems were removed, washed in running
water, dehydrated and embedded in paraffin. The brainstems from the four animals
in the initial experiment were embedded
in the same paraffin block. The brainstems
from the two or three animals killed at
each time interval after injury in the second experiment were likewise embedded
in a single paraffin block. The pieces of
brain stem were all oriented in the same
manner, and a piece of gut from one of
the animals, which served as a marker and
as a control on the radioautographic pro-
cedure, was placed to their left sides. Sections 7 u thick were cut throughout the
region containing the hypoglossal nuclei.
After the sections were mounted and the
paraffin was removed, the slides were
stained by the periodic acid-Schiff technique, dried and then coated in the darkroom with Kodak NTB-2 emulsicin. After
the emulsion had dried, the slides were
placed in lightproof Bakelite boxes containing a drying agent and were allowed
to expose at 4" C for 8-13 weeks. The
slides were than developed in Kodak
Microdol-X and later were stained with
hematoxylin, dehydrated, and mounted in
Permount.
The sections of medulla from the four
animals in the initial experiment were
studied in the following way. The sections
on every other slide (5-8 sections per
slide) extending from the level of the obex
to the decussation of the pyramids were
numbered in sequence. A table of random
numbers was then used to select five sections from each animal for study. The area
analyzed on each of these sections was
made up of two adjacent squares 0.3 mm
on a side with the common side in the
midline and with the dorsal side passing
through the central canal (fig. 2 ) . These
squares were found to contain most of the
neurons of the hypoglossal nucleus in the
section. Using an ocular grid, the sections
were scanned under oil immersion for
labeled cells and for mitotic figures. Background labeling was determined for each
slide studied by counting the number of
silver grains in ten randomly selected
areas of 2500 uz each, not containing tissue. The level of background ranged from
0.2 to 1.8 silver grains per 100 pz. Nuclei
overlain by four or more silver grains were
considered to be labeled and were marked
on photographs of the section being
scanned. The number of labeled cells, the
number of silver grains per labeled nucleus, and the number of labeled and unlabeled mitotic figures found in each 0.09
mmz area were recorded. From each animal
one additional section from the level of the
caudal end of the fourth ventricle was
studied. The number of hypoglossal neurons and the total number of non-neuronal
nuclei (excluding endothelial and ependy-
101
LEUCOCYTES IN THE HYPOGLOSSAL NUCLEUS
ma1 cells) in each 0.09 mm2 were recorded
for each of these sections.
The material from the 11 animals in
the second experiment was analyzed as
follows. From each animal the section
nearest the point where the fourth ventricle meets the central canal was selected
for study. This region was selected because
it was identifiable and because it was near
the longitudinal midpoint of the hypoglossal nuclei. These sections were analyzed
in the manner already described. In addition the entire area of each of these sections was scanned under oil immersion for
labeled cells and mitotic figures. In the
animals killed on the fourth day after injury every twentieth section from the
decussation of the pyramids rostrally
throughout the length of the hypoglossal
nucleus was scanned under oil immersion
for labeled cells and mitotic figures, and
such cells were marked on photographs of
the section. The grain counts of all labeled
cells were also recorded. On the photographs of all the sections studied squares
that were 0.3 mm, 0.4 mm, 0.5 mm and
0.6 mm on a side were drawn (fig. 1 3 ) .
The total number of labeled cells contained in each of these square areas (0.09
mm2, 0.16 mm2, 0.25 mm2, 0.36 mm2) on
either side of the midline was recorded.
RESULTS
In most of the sections studied from the
injured animals, the neurons of the left
hypoglossal nucleus had a noticeably increased cytoplasmic basophilia compared
to those of the right hypoglossal nucleus
(fig. 2). The number of labeled cells found
in the 0.09 mm2 area on both sides of each
section from the four animals in the initial
experiment is shown in table 1. There were
211 labeled cells on the left side and 36
labeled cells on the right side of the 20
sections studied. Using the technique of
analysis of variance as described by Dixon
and Massey ('57), it was shown that the
mean number of labeled cells per section
in the left hypoglossal nucleus was significantly greater ( p < 0.01) than that in
the right hypoglossal nucleus. Table 2
shows the number of neurons and the
number of non-neuronal nuclei (both labeled and unlabeled) found per 0.09 mmz
in one section from each of the four animals. There was no significant difference
in the number of nerve cells in the right
and left hypoglossal nuclei. The number
of non-neuronal nuclei per 0.09 mmz
ranged from 97 to 115 on the left side and
from 71 to 87 on the right side, with the
difference in means between the two sides
being significant ( p < 0.01). The ratio of
TABLE 1
Comparison o f the number o f labeled cells in 7 p sections containing the left and right
hypoglossal nuclei four days following injury to the left hypoglossal nerve. Three injections of
2 pc per gram body weight of tritiated thymidine were given during the day before the injury,
the last at 16 hours before injury. Each number represents the number o f labeled cells in
0.09 mm?.
Animal
Left
1
2
3
4
11
10
12
10
15
11
2
9
14
23
13
5
6
13
14
13
12
4
7
-
7
-
-
-
Total
44
50
64
53
2
1
4
2
0
3
Right
2
1
2
0
2
3
1
2
1
4
Total
3
1
2
0
-
-
-
9
9
9
Total
211
-
36
9
-
247
Mean number of labeled cells per 0.09 mm2
Left 10.6 & 4.7
Right 1.8t 1.2
p
< 0.01
102
E. K. ADRIAN, JR. AND R. D. SMOTHERMBN
TABLE 2
Cell population o f the left ( L ) and right ( R ) hypoglossal nuclei i n the section at the caudal
end of the fourth ventricle. The areas considered are each 0.09 mm2
Neurons
L
1
2
3
4
Mean
1
36
26
27
33
30.5
Non-neuronal Nuclei
R
34
23
24
33
28.5
97
115
103
106
105.3
p
0.01
<
78
71
81
87
79.3
Non-neuron/Neuron
L
2.69
4.42
3.81
3.21
3.53
p
0.01
<
R
2.29
3.09
3.38
2.65
2.85
Endothelial and ependymal nuclei excluded.
non-neuronal cells to neurons was also significantly higher on the left side when the
data were paired ( p < 0.01).
The labeled nuclei on both sides were
for the most part dark staining and elongated or irregular in shape, and most of
them were weakly labeled (fig. 3 ) . Of the
247 labeled cells observed, only five had
more than 25 grains over their nuclei. Four
of these heavily labeled cells were on the
left side. The mean grain count of the
remaihing 207 cells on the left side was
6.0 1.7. There was no significant difference in the mean grain count between the
two sides, but there was an extremely low
probability ( p < 0.01) that cells with grain
counts greater than 25 belonged to the
same population as the other labeled cells.
The results of the second experiment are
shown in tables 3,4,and 5. Table 3 shows
the number of labeled cells found in the
three animals that were sacrificed five days
after receiving three injections of 4 wc tritiated thymidine per gm body weight (four
days after nerve injury). The mean number of labeled cells per section was from
25 to 40 times higher in the animals that
had been subjected to hypoglossal nerve
injury than in the uninjured control. The
number of labeled cells in the areas studied in the right hypoglossal nucleus of the
injured animals was from four to seven
times higher than the number of labeled
cells in the right hypoglossal nucleus of
the uninjured control. The number of labeled cells in the left hypoglossal nucleus
of the injured animals was 5 to 12
times higher than the number of labeled
cells in the right hypoglossal nucleus of
the same animals. Labeled cells were found
on both sides in a larger area than the
*
1
R
L
0.3 mm by 0.3 mm square studied in the
initial experiment, although most of the
hypoglossal neurons were in the smallest
(0.09 mm2) square. However, the relative
distribution of labeled cells between the
two sides showed little change when the
larger squares were considered. Labeled
cells were rare outside the limits of the
0.6 mm by 0.6 mm square on either side
in both the injured animals and the uninjured control. There was no concentration of labeled cells associated with the
exiting fibers of the hypoglossal nerve. Labeled cells were present throughout the
length of the nucleus of the injured hypoglossal nerve, but at either end of the nucleus the number of such cells was very
small.
Table 4 shows the number of labeled
cells found in one section at the level of
the rostra1 end of the central canal from
each of the eleven animals in the second
experiment. Only three labeled cells were
found in the squares containing the hypoglossal nuclei in the sections from the four
uninjured control animals. The sections
from the injured animals, on the other
hand, had a much greater number of labeled cells at all time intervals after injury
than did the corresponding uninjured controls. The mean number of labeled cells
per section in the right hypoglossal nucleus
of the injured animals was significantly
greater than that found in the uninjured
controls, regardless of the area considered
( p < 0.01). The mean number of labeled
cells per section on the left side of the injured animals was significantly higher than
that on the right side ( p < 0.01). The
largest number of labeled cells was found
103
LEUCOCYTES IN THE HYPOGLOSSAL N U C L E U S
TABLE 3
Number o f labeled cells in 7 p sections containing the left ( L ) and right ( R ) hypoglossal
nuclei. Adjacent squares w i t h their common side i n the midline and w i t h areas as shown
below were scanned under oil immersion for labeled cells. Each animal received three injections o f 4 ,uc per g m body weight o f tritiated thymidine. The left hypoglossal nerve was cut
one day later i n animals N o . 6 and N o . 7 . All three animals were sacrificed on the f i f t h day
after the injections o f tritiated thymidine (four days after injury).
Number of labeled cells
Animal
No. 5
(Control)
No. 6
0.09 mm2
0.16 mm2
0.25 mm2
0.36 mm2
L
R
L
R
L
R
L
R
0
0
1
0
0
1
0
0
0
0
2
0
1
2
1
0
0
0
1
0
2
0
1
2
1
0
0
0
1
0
2
0
1
3
0.6
3
0.6
4
0.8
3
0.6
4
0.8
-___
0
1
2
1
0
0
0
1
0.2
2
0.4
3
0.6
5
3
17
24
20
69
13.8
No. 7
3
0
5
3
4
14
18
25
34
23
3
4
6
4
5
17
24
27
39
30
5
4
6
4
5
20
27
32
42
31
5
4
6
4
5
15
114
22.8
22
4.4
137
27.4
24
4.8
152
30.4
24
4.8
5
5
7
2
1
4
19
4.0
3.0
29
18
25
21
22
2
3
1
1
3
39
36
39
27
24
5
4
1
1
3
46
46
47
33
25
2
1
4
51
52
49
34
25
115
23.0
10
2.0
165
33.0
14
2.8
197
39.4
19
3.8
211
42.2
on the eighth day after injury. The number
of labeled cells had decreased considerably by the thirty-fifth day after injury, and
the difference between the two sides had
become much less marked. In all the sections studied labeled cells were present in
only small numbers ( 3 to 4 per mm2) outside the limits of the 0.6 mm by 0.6 mm
squares, and there was no significant difference between the injured animals and
the controls, No labeled cells were observed
along the intramedullary course of the injured nerves.
The total number of non-neuronal cells
per 0.09 mmz in the left hypoglossal nucleus of the injured animals was considerably increased over that found in the right
hypoglossal nucleus of the same animals
and over the number in the uninjured controls (fig. 1 ) . On the fourth, eighth, and
sixteenth days after injury, a substantial
7
Total
Mean
Total
Mean
Total
Mean
part of this increased number of cells was
labeled (dotted part of the bars in fig. 1).
By the thirty-fifth day after injury, although the total number of cells remained
above control levels, the proportion of them
that were labeled had decreased.
Although the number of labeled cells in
the nucleus of the injured hypoglossal
nerve increased between the fourth and the
eighth days after injury and decreased
thereafter, the mean grain count of the
labeled cells showed no significant change
(table 5). There was no significant difference in the mean grain count between the
two sides at any of the time intervals after
injury, nor was there a significant change
in mean grain count with time after injury.
Many of the labeled cells were located
in the typical position of “satellite” oligodendroglia or microglia cells (figs. 3, 4,
5, 6, 7 , 8, 14, 1 5 ) . With the hematoxylin
104
E . K. ADRIAN, JR. AND R. D. SMOTHERMON
TABLE 4
Number o f labeled cells i n the hypoglossal nuclei at the caudal end o f the fourth ventricle
Number of labeled cells
Day
0.09 mme
L
4
8
16
35
Total
Mean
0
0
0
0
0
0
0.25 mm2
0.16 mm2
R
R
L
L
Uninjured controls
0
0
0
0
0
0
0
1
0
1
0
1
1
1
1
0.25
0.25
0.25
0
0
1
0
1
0.25
0.36 mm2
R
L
0
0
0
1
0
1
0.25
0
0
1
1
0.25
-
R
0
0
2
0
2
0.50
Injured animals
4
8
16
35
12
26
32
19
25
6
4
4
4
18
39
54
49
45
12
11
1
5
4
6
4
26
47
65
71
57
15
11
6
1
6
6
8
4
4
27
49
6
2
6
9
6
2
a
60
a
a
5
5
16
11
5
5
69
78
10
100
Number o f
Non-Neuronal
Cells
50
0
C
C
8
4
Days
After
C
16
35
Injury
Fig. 1 The bars indicate the total number of non-neuronal nuclei (excluding the nuclei
of endothelial and ependymal cells) in a 0.09 mni2 area including most of the neurons of
the left ( L ) or the right ( R ) hypoglossal nucleus in a section at the level of the caudal
end of the fourth ventricle. The dotted portions of the bars represent labeled cells. Uninjured control animals are indicated by “C.”
stain used, there was nothing about the
shape or staining characteristics of their
nuclei that would allow them to be distinguished from such cells. An occasional
labeled cell had a larger light-staining nucleus (fig. l l ) , but such cells were rare.
Many labeled nuclei were located just outside vessel walls on the eighth day after
injury (fig. 9) and by the sixteenth day
after injury there were occasionally clusters of labeled cells around vessels (fig. 10).
At eight days after injury, some of the
neurons of the injured hypoglossal nucleus
were pyknotic and had numerous labeled
satellites (figs. 13, 14, 15).
Mitotic figures were present at four days
after injury (fig. 12), but were not found
at the later time intervals or in the uninjured controls. Of the thirteen mitotic
figures found, eight were labeled and five
were unlabeled. Eleven of the thirteen were
found in the injured hypoglossal nucleus,
105
LEUCOCYTES IN THE HYPOGLOSSAL NUCLEUS
TABLE 5
Mean grain count o f the labeled cells i n the animals that received three injections
of 4 U C tritiated thymidine per gram body weight
Exposure time
Day
after
injury
8 Weeks
R
4
8.6f4.7
8
16
8.4k3.5
7.8 & 2.9
6.6k2.5
35
13 Weeks
9% Weeks
L
L
R
L
8.9f4.1
9.5 t 3.6
10.1t4.6
8.7k4.1
R
10.1k4.7
9.9 k 2.6
8.2*2.9
8.2k2.9
method of tracing labeled leucocytes into
tissues having, normally, a low rate of
DNA synthesis has considerable precedent
DISCUSSION
(Bintliff and Walker, '60; Adrian and
Tritiated thymidine is generally consid- Walker, '62; Walker, '63; Konigsmark and
ered to be a specific precursor of DNA, and Sidman, '63; Huntington and Terry, '66;
nuclear labeling following its injection is Smith and Walker, '67; Adrian, '68; Olsson
a specific manifestation of DNA synthe- and Sjostrand, '69).
The labeled cells observed in this study
sized during the relatively short period in
which the thymidine is available following were not confined to vessels, since the aniinjection. This period of time probably does mals were perfused and the vessels were
not exceed one to two hours (Hughes et al., washed clear of cells. The walls of the ves'58). In this experiment three injections sels, which were found in all cases to be
of tritiated thymidine were given 24, 20, distended, were well stained with the peand 16 hours before injury of the left hy- riodic acid-Schiff technique. Although frepoglossal nerve. Accordingly, there should quently found in direct relationship to
have been no isotope available to label cells vessel walls (figs. 9, l o ) , many of the lawhich proliferated in response to the in- beled cells were also found close to neurojury, although such proliferation is well nal perikarya (figs. 3, 4, 5, 6, 7, 8, 14,
known to occur (Cammermeyer, '65a,b; 15), and there was nothing about their
Sjostrand, '65a,b).
shape or staining characteristics that would
The left hypoglossal nucleus in the sec- distinguish these cells from the cells fretions studied from the injured animals was quently referred to as perineuronal satelfound to have a much greater number of lites.
In earlier studies where mononuclear
labeled cells than the nucleus of the uninjured nerve. However, the right hypo- leucocytes have been identified in nervous
glossal nucleus of the injured animals had tissue by means of tritiated thymidine lasignificantly more labeled cells than were beling (Adrian and Walker, '62; Walker,
found in the uninjured controls. No evi- '63; Konigsmark and Sidman, '63; Huntdence was found that the small number of ington and Terry, '66; Smith and Walker,
labeled cells normally present in the ner- '67: Adrian, ' 6 8 ) , there has been some type
vous tissue following tritiated thymidine of local lesion to the nervous tissue in
administration migrated to the injured hy- which vessel walls have been disrupted.
poglossal nucleus or underwent selective Thus, it has been impossible to prove diproliferation there. Since there was no large rect passage of the labeled leucocytes
population of labeled cells in or near the through intact vessel walls. In this study
hypoglossal nucleus, except those which the only disruption of vessels oqcurred
were in the blood, it seems very likely that when the hypoglossal nerve was dissected
most of the labeled cells observed in the out and divided at a considerable dfstance
injured animals were blood-derived. The from the site where the labeled cells were
weak label found over most of these cells observed. Therefore, it seems likely that
is consistent with this interpretation. This mononuclear leucocytes are able to pass
while two were found in the nucleus of
the uninjured nerve.
106
E. K. ADRIAN, JR. AND R. D. SMOTHERMON
directly through uninjured vascular walls
in nervous tissue, as they do in other tissues. The decreased number of labeled
cells after eight days without a corresponding decrease in grain count would be consistent with their migrating out again.
While migration of labeled cells proximally
along the central part of the injured nerve
may possibly occur, the evidence at hand
does not lend support to this hypothesis.
Labeled cells were not observed in association with the intramedullary fibers of
the injured hypoglossal nerve in this experiment. In a similar study where the
sciatic nerve was injured following labeling of the hemopoietic tissues with tritiated thymidine, relatively few labeled
cells were found in the central part of the
injured nerve (Olsson and Sjostrand, '69).
It should be noted that more than half
of the observed mitotic figures were labeled. While there was approximately a
30% increase in the number of non-neuronal cells in the injured hypoglossal nuclei, about one-third to one-half of this
increase can be accounted for directly by
the presence of the labeled cells. Since only
a fraction of the migratory cells would be
labeled, they must be responsible for a substantial portion of the observed increase in
non-neuronal cells and for much of the
observed mitotic activity. The functional
significance of this infiltration and the
morphology of these cells using the electron microscope and other techniques are
presently under study.
LITERATURE CITED
Adrian, E. K., and B. E. Walker 1962 Incorporation of thymidine-H3 by cells i n normal
and injured mouse spinal cord. J. Neuropath.
Exp. Neurol., 21: 597-609.
Adrian, E. K. 1968 Cell division in injured
spinal cord. Am. J. Anat., 123: 501-520.
Bintliff, S., and B. E. Walker 1960 Radioautographic study of skeletal muscle regeneration.
Am. J. Anat., 106: 233-246.
Cammermeyer, J. 1965a Juxtavascular karyokinesis and microglia cell proliferation during
retrograde reaction in the mouse facial nucleus.
Ergebn. Anat. Entwicklungsgesch., 38: 1-22.
- 1965b Histiocytes, iuxtavascular mitotic
cells and microglia cells during retrograde
changes in the facial nucleus of rabbits of
varying age. Ergehn. Anat. Entwicklungsgesch.,
38: 195-229.
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PLATES
PLATE 1
EXPLANATION OF FIGURES
2
Cross section of medulla of one of the four animals in the initial
experiment. The two adjacent squares are each 0.3 m m on a side.
The square on the right encloses the nucleus of the injured left hypoglossal nerve. Arrows point to radioactive cells. The neurons in the
injured nucleus had a more pronounced cytoplasmic basophilia than
did those in the nucleus of the uninjured nerve. Periodic acid-Schiff
hematoxylin, x 185.
3 Arrows point to labeled nuclei, each of which is i n close proximity
to a neuron in the nucleus of the injured hypoglossal nerve. Four
days after injury. PAS-hematoxylin, X 1150.
4
108
Several labeled nuclei are indicated, most of which are in a position
satellite to a nerve cell in the nucleus of the injured hypoglossal
nerve. Eight days after injury. PAS-hematoxylin, x 1300.
LEUCOCYTES IN THE HYPOGLOSSAL NUCLEUS
E. K. Adrian. Jr. and R. D. Smothermon
PLATE 1
109
PLATE 2
EXPLANATION OF FIGURES
110
5
Neuron i n nucleus of injured hypoglossal nerve with three labeled
satellites. Eight days after injury. Focused on silver grains. PAShematoxglin, X 1300.
6
Same as figure 5, but focused on nuclei.
7
Labeled perineuronal satellite in nucleus of injured nerve. Eight days
after injury. Focused on silver grains. PAS-hematoxylin, X 1300.
8
Same as figure 6, but focused on nucleus,
LEUCOCYTES IN THE HYPOGLOSSAL NUCLEUS
E. K. Adrian, Jr. and R. D. Smothermon
PLATE 2
111
PLATE 3
EXPLANATION OF FIGURES
9
Arrows indicate four labeled cells, three of which are located just
outside capillary walls. Eight days after injury. PAS-hematoxylin,
x 1300.
10 Six labeled cells clustered around a capillary are indicated in the
lower right corner of the picture. A single labeled cell just outside
a capillary is shown in the upper left corner. Sixteen days after
injury. PAS-hematoxylin, >< 1300.
11
A labeled light-staining nucleus is indicated by the arrow. Such
cells were extremely rare. Sixteen days after injury. PAS-hematoxylin,
x 1350.
12 The arrow indicates a mitotic figure closely adjacent to a neuron in
the nucleus of the injured hypoglossal nerve. Four days after injury.
PAS-hematoxylin, x 1150.
112
LEUCOCYTES IN THE HYPOGLOSSAL NUCLEUS
E. K. Adrian, Jr. and R. D. Smothermon
PLATE 3
113
PLATE 4
EXPLANATION OF FIGURES
13 Cross section of medulla a t the level where the central canal begins
to open to form the fourth ventricle. The nucleus of the injured
hypoglossal nerve lies to the right in the picture. A line 0.6 mm long
has been drawn in the midline ventrally from the most ventral point
of the lumen of the central canal. Squares 0.3 mm, 0.4 mm, 0.5 mm,
and 0.6 mm on a side have been constructed with a common corner
in the central canal. The arrows indicate labeled cells. A number of
dark staining, pyknotic neurons can be seen in the nucleus of the
injured nerve, and a smaller number of such cells are also visible
in the opposite hypoglossal nucleus. Eight days after injury. PAShematoxylin, X 115.
114
14
An enlarged view of one of the dark-staining neurons shown above.
Arrows indicate four labeled satellites. Focused on silver grains. PAShematoxylin, x 1300.
15
Same as figure 14, but focused on nuclei.
LEUCOCYTES IN THE HYPOGLOSSAL NUCLEUS
E. K. Adrian, Jr. a n d R. D. Smothermon
PLATE 4
115
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